Diagnostic ultrasound imaging is based on the principle that sound waves can be focused upon an area of interest and reflected in such a way as to produce an image thereof. The ultrasonic transducer is placed on a body surface overlying the area to be imaged, and ultrasonic energy in the form of sound waves is directed toward that area. As ultrasonic energy travels through the body, the velocity of the energy and acoustic properties of the body tissue and substances encountered by the energy determine the degree of absorption, scattering, transmission and reflection of the ultrasonic energy. The transducer then detects the amount and characteristics of the reflected ultrasonic energy and translates the data into images.
As ultrasound waves move through one substance to another there is some degree of reflection at the interface. The degree of reflection is related to the acoustic properties of the substances defining the interface. If these acoustic properties differ, such as with liquid-solid or liquid-gas interfaces, the degree of reflection is enhanced. For this reason, gas-containing contrast agents are particularly efficient at reflecting ultrasound waves. Thus, such contrast agents intensify the degree of reflectivity of substances encountered and enhance the definition of ultrasonic images.
Ophir and Parker describe two types of gas-containing imaging agents: (1) free gas bubbles; and (2) encapsulated gas bubbles (Ultrasound in Medicine and Biology 15(4):319-333 (1989)), the latter having been developed in an attempt to overcome instability and toxicity problems encountered using the former. Encapsulated gas microbubbles, hereinafter referred to as "microspheres," are composed of a microbubble of gas surrounded by a discrete shell of protein or other biocompatible material. Two such protein-shelled imaging agents are ALBUNEX.RTM., which consists of a suspension of air-containing albumin microspheres, and OPTISON.TM., which consists of a suspension of perfluoropropane-containing albumin microspheres (both of Molecular Biosystems, Inc., San Diego, Calif.). Other examples of microspheres include surfactant coated protein microspheres (Giddey, WO 92/05806), covalently-crosslinked protein microspheres (Feinstein et al., U.S. Pat. Nos. 4,718,433 and 4,774,958; and Klaveness et al., U.S. Pat. No. 5,529,766); and microspheres with biodegradable synthetic polymer shells (Rossling, et al., U.S. Pat. No. 5,501,863; and Bernstein et al., U.S. Pat. No. 5,611,344).
Microspheres are part of a broader category of contrast agents referred to herein as "microbubble-based contrast agents", or simply "microbubble contrast agents." These types of agents derive at least part of their ability to provide contrast by being capable of supplying a plurality of gaseous microbubbles to the site of imaging. In addition to the free gas microbubbles and encapsulated gas microbubbles described by Ophir and Parker, supra, this class of imaging agents includes emulsions containing chemicals that are in the gaseous state or are capable of becoming gaseous (hereinafter collectively referred to as "gaseous emulsions") prior to or during the application of ultrasound.
A variety of different mechanisms have been utilized to enhance the ability of gas-generating emulsions of volatile liquids to serve as ultrasound contrast agents. One mechanism involves the ability of emulsions of volatile liquids to be stabilized in the liquid state until ultrasound energy is applied at the imaging site, which induces vaporization of the liquid to form microbubbles (U.S. Pat. No. 5,536,489). Another mechanism involves the use of volatile liquids which undergo a phase shift from liquid to gas in vivo upon an increase in temperature to body temperature (U.S. Pat. Nos. 5,558,853 and 5,558,854). Still another mechanism involves the ability to promote microbubble formation in vitro prior to application of the contrast agent by subjecting it to a decrease in pressure (PCT WO 96/40282).
Another type of microbubble contrast agent utilizes solid particles which are capable of carrying gas microbubbles on their surface, or form gas microbubbles upon dissolution in a carrier liquid and/or the bloodstream. See for example, U.S. Pat. Nos. 5,147,631; 4,265,251; 4,657,756; and Australian Patent No. 89/40651.
The first generation of microbubble contrast agents generally involved the use of soluble gases, such as air and nitrogen. However, these contrast agents were shown to be of limited use, because their instability in vivo results in a rapid loss of echogenicity following injection. The instability of these agents necessitates repeated and continuous dosing, which is generally undesirable because of the increased volume of injected gas, as well as the added cost.
Attempts at stabilizing microbubble imaging agents has recently focused on the use of less soluble gases. For example, U.S. Pat. No. 5,413,774 describes the use of at least a portion of gas that has a S.sub.gas /.sqroot.MW.sub.gas .ltoreq.0.0031, where S.sub.gas is the water solubility of the gas and MW.sub.gas is the average molecular weight of the gas. Also, U.S. Pat. No. 5,529,766 describes the use of protein microbubbles using low molecular weight fluorinated hydrocarbons or sulfur hexafluoride; and U.S. Pat. No. 5,573,751 describes the use of a variety of different fluorine-containing gases.
Stabilization of microbubble contrast agents has greatly improved their overall efficacy for certain applications. For example, one type of ultrasound imaging study involves the use of microbubble contrast agents to trace the flow of blood through the microcirculation. This type of study relies on the ability of certain types of contrast agents, called "tracer agents," to mimic the flow of blood cells through the microcirculation, and also requires that they be stable enough to survive the transit through the microcirculation. However, this application is not easily performed with microbubbles that are stable enough to survive multiple passes through the microcirculatory system under investigation. This is because the study of microcapillary circulation depends on the ability to determine the echogenicity of the microcapillaries during the first pass of the contrast. Hence, accumulation of the contrast agent with each pass makes it difficult to determine the relationship between echogenicity after administration and the amount of contrast agent which was administered.
In addition to having increased in vivo persistence, certain stabilized contrast agents also have a tendency to become preferentially lodged in various tissues or in the microcirculation. These types of contrast agents are sometimes called "depot agents." Examples of depot agents include: Quantison Depot.RTM., (Andaris, Nottingham, England); and EchoGen.RTM., (Sonus, Bothell, Washington; See Journal of the American Society of Echocardiography, 10(1):11-24 (1997)). Although less useful for studying blood flow, depot agents can thus have the added benefit of localized enhancement of ultrasound images. For this reason, many ultrasound contrast agents are designed specifically as depot agents through the use of targeting moieties which cause them to become concentrated at a desired imaging site. See, for example, PCT WO 94/08627. However, such targeted contrast agents can have the disadvantage of maintaining persistent echogenicity at the target site for such a long duration that their prolonged presence interferes with the ability to perform subsequent imaging studies with a second dose of contrast agent (i.e. a multiple dose imaging study). Furthermore, depot agents may exhibit toxic side effects if they obstruct the microcirculation for an undue length of time.
It has recently been reported that prolonged exposure to ultrasound energy can rapidly diminish the echogenicity of microbubble contrast agents after administration (Vandenberg and Melton, J. Am. Soc. Echocardiogr. 7:582-589 (1994)). In an attempt to circumvent this problem, Porter has described that the destructive effects of ultrasound can be avoided by limiting the exposure of the microbubble contrast agents to ultrasound energy after injection (U.S. Pat. No. 5,560,364). Although for most applications, the effects of exposure to ultrasound energy are considered to be undesirable, it has previously been reported that for at least one type of imaging study, the effects of ultrasound energy on echogenicity can be used beneficially. In particular, it was reported that during an imaging study of the myocardium involving a continuous venous infusion (a single dose imaging study) of a microbubble imaging agent, application of ultrasound energy to the myocardium can be used to temporarily eliminate echogenicity of the contrast agent in order to assess reperfusion of the contrast agent back into the myocardium (Wei et al., JACC 29(2):1081-1088 (1997)). However, previous reports have not been identified that describe the use of increased exposure of microbubble contrast agents to ultrasound energy to facilitate multiple dose imaging studies.
Many different imaging studies call for the administration of two or more doses of ultrasound contrast agent. For example, when a patient is undergoing a stress echocardiogram, whether pharmacological agents or mechanical exercise is used as a stressor, it is necessary to initially obtain an ultrasound image of the patient at rest, in the presence of contrast agent, then subject the patient to the stress, and obtain a second diagnostic image or images at peak stress. These studies are designed to assess such conditions as reversible ischemia, exercise induced angina or unstable angina. Stress echocardiography employs either no ultrasound contrast agents or ALBUNEX.RTM. contrast agent (Molecular Biosystems, Inc., San Diego, Calif.) for enhancing the assessment of wall motion defects that follow myocardial ischemia Currently, several ultrasound contrast agents are in clinical trials for use in a method of assessing myocardial perfusion status. In these studies, ultrasound is used after injection of a contrast agent at rest, followed by an inducement of stress, and then by a second injection of contrast agent during peak stress. Because the "at rest" and "peak stress" injections are often given very close together, there may be an interference in the myocardial echogenicity after the second dose due to persistent echogenicity from the first dose. This results in the inability to obtain useful information from the second injection.
As described above, the persistence of echogenicity from a first dose of a microbubble contrast agent can interfere with subsequent imaging studies utilizing a second dose. Such interference of residual ultrasound tissue enhancement from previous doses of contrast agents could be described as a nuisance or a confounding factor in the accurate assessment of changes in tissue perfusion status over the course of a study. The researcher or clinician, using visual assessments of gray scale brightness or more quantitative methods of densitometry, may not be able to discern ultrasound contrast in the tissue as resulting from pre or from post intervention for example.
It is therefore an object of the present invention to provide a method of imaging which allows for a first dose of a microbubble contrast agent to be quickly and effectively "cleared" from the imaging field so that a second dose can be administered and a second imaging study performed which is free from interference due to the persistence of the first dose. This allows clinicians to use echogenically persistent microbubble contrast agents to enhance ultrasound intensity without the associated interference with subsequent imaging studies after additional administration of contrast agent. Utilizing this method, the clinician is able to capture ultrasound images derived from a first administration of contrast agent during any phase of its echogenic effect on the imaging site. After completion of the initial imaging study, the clinician is able to efficiently eliminate residual echogenicity so that subsequent imaging studies which may follow surgical intervention or treatment can be immediately performed without interference.